Voltage-gated Ca2+ channels are important regulators of membrane potential in cardiac, skeletal and smooth muscle cells and cardiovascular neurons. Just as important is the Ca2+ the enters via these channels acts as a second messenger upon ion channels, C kinase, proteases, calmodulin, etc. The structure of Ca2+ channels in skeletal muscle is tetrameric consisting of alpha 1, alpha2, beta, and gamma subunits; elsewhere the subunit composition is unknown. We showed that this alpha1 subunit by itself can function as a Ca2+ channel and dihydropyridine receptor (Perez-Reyes et al., 1990). This was achieved by stably transfecting alpha1 into murine L cells which are devoid of all subunits and so not express Ca2+ currents. Mikami et al. (1990) showed that the alpha1 subunit from cardiac muscle expressed Ca2+ currents in Xenopus oocytes. Since oocytes have an endogenous Ca2+ current, the properties of alpha 1 were not specified as clearly. The molecular mechanisms of voltage sensitivity, divalent cation selectivity and regulation of Ca2+ channels are largely unknown. We will examine these properties for both alpha 1 subunits in combination with different skeletal muscle alpha 2, beta, gamma subunits and different cardiac muscle subunits as they become available. Expression will be studied in L cells and oocytes. Chimeras of the two alpha 1s will be used to determine the repeats responsible for the differences between skeletal and cardiac muscle Ca2+ currents. Chimeras with Na+ and K+ channels will be developed to determine the repeats important for gating and selectivity. The search for the essential parts will be narrowed by using segments from candidate repeats. These will be introduced into a K+ channel which has been genetically engineered for segment swapping. Sites for regulation by G proteins will be tested using similar approaches.